TUMOR SUPPRESSOR PROTEIN PRB2, RELATED GENE PRODUCTS, AND DNA ENCODING THEREFOR

Abstract: The invention provides a novel tumor suppressor protein (pRb2) of the retinoblastoma family, and DNA encoding therefor is provided.

Description: TUMOR SUPPRESSOR PROTEIN PRB2, RELATED GENE PRODUCTS, AND DNA ENCODING THEREFOR Field of the Invention This invention relates to a tumor suppressor protein (pRb2) of the retinoblastoma family which binds to the E1A transforming domain. The invention also concerns DNA encoding for pRb2 and related gene products. Background of the Invention Many types of human cancer are now believed to be caused by an imbalance of growth regulators within a cell. A decrease in negative control growth regulators and/or their deactivation can cause a cancerous con- dition. Further, an increase in positive control growth regulators can also cause a cancerous condition. Since the identification of the first tumor suppressor gene, much effort in cancer research has been focused on the identification of new tumor suppressor genes and their involvement in human cancer. Many types of human cancers are thought to develop by a loss of heterozygosity of putative tumor suppressor genes not yet identified (Lasko et al., Annu. Rev. Genetics. 25, 281- 296 (1991)) according to Knudson's "two-hit" hypothesis (Knudson, Proc. Natl . Acad. Sci. USA. 68, 820-823 (1971)) . One of the most studied tumor suppressor genes is the retinoblastoma susceptibility gene (rb) , whose gene product (pRb) has been shown to play a key role in the regulation of cell division. In interphasic cells, pRb contributes to maintaining the quiescent state of the cell by repressing transcription of genes required for the cell cycle through interaction with transcription factors, such as E2F (Wagner et al .. Nature, 352 , 189-190 (1991) ; Nevins, Science. 258. 424-429 (1992) ; and Hiebert et al. , Genes Develop. , 6_. 177-185 (1992)) . The loss of this activity can induce cell transformation as evidenced by the reversion of the transformed phenotype in pRb cells after replacement of a functional pRb (Huang et al. , Science 242 1563-1565 (1988) ; Bookstein et al . , Science, 247 712-715 (1990) ; and Sumegi ____________________, Cell Growth Differ.. 247-250 (1990)) . Upon entrance into the cell cycle, pRb seems to be phosphorylated by cell cycle-dependent kinases (Lees et al.. EMBO J. 10 4279-4290 (1991) ; Hu et al . , Mol. Cell. Biol.. 12 971-980 (1992); Hinds et al. , Cell. 70 993-1006 (1992) ; Matsushime et al.. Nature. 35 295- 300 (1992)) which is thought to permit its dissociation from transcription factors and, hence, the expression of genes required for progression through the cell cycle. Noteworthily, the association of pRb with cell cycle regulators like cyclins and cell cycle-dependent kinases suggests a universal character to its function. However, pRb involvement in human cancer has been restricted to a limited number of tumor types suggesting that this hypothetically universal function may be exerted by other gene products in a cell type- specific manner. Consistently, knock out of the rb gene in mice affects only specific cell types and after sever¬ al days of embryonic development (Jacks et al. , Proc. Natl. Acad. Sci . USA. 68 820-823 (1992) ; Lee et al .. Nature, 359 288-294 (1992) ; Clarke et al . , Nature. 359 328-330 (1992) ) . The ability of several transforming proteins from human DNA tumor viruses to activate cell proliferation has been a useful tool for the identi¬ fication of cellular factors involved in the regulation of the cell cycle. Negative regulators of cell growth may thus be effective targets for inactivation by these viral proteins, as it occurs with the product of the retinoblastoma gene. Adenovirus E1A, SV40 T antigen, and papilloma- virus E7 are three viral proteins which have been found to bind to pRb. This binding is responsible for the release of transcription factors required for the expression of cell cycle genes (Nevins, Science, 258424- 429 (1992) ; Bandara et al.. Nature, 351 494-497 (1991)) . A conserved motif found in the three viral proteins allows for interaction and complex formation with pRb (Moran, Curr. Op. Gen. Dev.. 3_ 63-70 (1993)) . In the case of the adenovirus E1A protein, this motif is located in the transforming domain 2, which is required for growth activation. The pRb-related product pl07 also binds in this region (Egan et al.. Mol . Cell . Biol. , 8 . 3955-3959 (1988) ; Whyte et al.. Cell. 5β_ 67-75 (1989)) . Domain 2 is also the site of interaction of an additional ElA-binding protein, pl30 (Giordano et al . , Oncoαene. 6. 481-485 (1991)) . This has led to the sug¬ gestion that pl30 has a structural relationship to pRb and pl07 (Moran, Curr. O . Gen. Dev.. 3 . 63-70 (1993)) . The ElA-binding domain in pRb and pl07 is a conserved region termed the "pocket region" (Kaelin et al.. Mol. Cell. Biol.. .10 3761-3769 (1990) ; Ewin et al..Cell. 66 1155-1164 (1991)) , and it is thought to play a primary role in the function of these proteins. The pocket is structurally formed by two regions A and B, which are conserved in pRb and pi07 and separated by non- conserved spacers of different sizes in pRb and pl07. In addition to pRb and pl07, there are other cellular ElA-binding proteins that have been identified by co-immunoprecipitation experiments using antibodies to E1A. These cellular proteins include the major polypeptides p300, pl30, p60/cyclin A, and several other minor forms (Yee, et al■ , Virology 147 142-153 (1985) ; Harlow et al. , Mol. Cell. Biol. 6 . 1579-1589 (1986) ; Giordano, et al. , Cell 58 981-990 (1989) ; Giordano et al . Science 253 1271-1275 (1991) ) . Binding to the N-terminal region has been shown to be exclusive to p300 (Egan et al.. Mol. Cell. Biol.. 8 3955-3959 (1988) ; Whyte et al. , Cell. 56 67-75 (1989) ; Stein et al.. J. Virol.. 64 4421- 4427 (1990)), and pRb2 consistently failed to bind to this region. Both domains 1 and 2 of the E1A protein have been shown to be necessary for the E1A binding of the following set of proteins: pRb, pl07, p60/cyclin A, and pl30 (Egan et al.. Mol. Cell. Biol.. 8. 3955-3959 (1988) ; Whyte et al.. Cell. 56 . 67-75 (1989) ; Giordano et al. Science 253 1271-1275 (1991)) . Furthermore, the E1A- 928 mutant has been previously shown to bind to pi07 and p60/cyclin A, but not to pRb and pl30. The association of pRb with transcription factors, such as E2F, occurs by interactions at the pocket region (Raychaudhuri et al.. Genes Develop.. 5 . 1200-1207 (1991)) and, recently, pl07 has also been shown to exert such a binding profile (Cao et al.. Nature, 355 176-179 (1992) ) . Moreover, the pocket region is found mutated in several human cancers where a lack of function of the pRb protein is thought to be involved in the ac¬ quisition of the transformed phenotype (Hu et al .. EMBO ___, 9_ 1147-1153 (1990)) ; Huang et al.. 1990) . There is a need for identification and sequenc- ing of new rb-related genes that may have an involvement in cell growth inhibition. Genes related to rb and their protein products that also have tumor suppressor activity in specific cell types are needed. Brief Description of the Figures Figure 1 is a schematic representation of wild type and mutant forms of the E1A protein. The mutant forms are pm928/961, dl2-36, dl38-67 and dl73-120. The nature of each mutation is set forth schematically. Figure 2 is an SDS-PAGE gel showing binding of the pRb2 protein to a fusion construct of a wild-type E1A protein and a glutathione-S-transferase (GST) protein (lane 2) and to E1A mutant constructs fused to a GST protein: dl2-36 (lane 3), dl38-67 (lane 4) , dl73-120 (lane 5) , and pm928/961 (lane 6) . GST protein with no E1A fused was included as a control (lane 1) , which showed no binding with the pRb2 protein. Summary of the Invention The present invention provides a recombinant DNA cloning vehicle comprising a DNA sequence comprising the human pRb2 gene DNA sequence. A preferred DNA se¬ quence is a sequence according to SEQ ID NO:l. The DNA comprises a sequence coding for the amino acid sequence according to SEQ ID NO:2. In another embodiment, the present invention provides a protein essentially having an amino acid se¬ quence according to SEQ ID N0:2. Preferably, the protein corresponding to SEQ ID NO:2 is not phosphorylated. In a further embodiment the present invention provides a host cell line transformed by the DNA of the cloning vehicle described above, which host cell line expresses the DNA from the cloning vehicle to produce a protein. Preferably the DNA has a sequence according to SEQ ID NO:l and the protein produced has a sequence ac- cording to SEQ ID NO:2. Detailed Description of the Invention The pRb2 protein is a previously unsequenced, uncharacterized member of the pRb family of tumor sup¬ pressor proteins. This protein was designated as pRb2 since it binds to Adenovirus E1A protein in a manner similar to pRb and pl07. Thus, the cDNA sequence coding for pRb2 was designated as the pRb2 gene. Polymerase chain reaction (PCR) using probes derived from domains 1 and 2 of the rb gene was utilized to identify the pRb2 cDNA sequence as follows. Synthetic degenerate oligonucleotides were designed based on conserved amino acid sequences flanking the spacers in pRb and pl07. These oligonucleotides were designated as primer A and primer B and used as PCR prim- ers to isolate and clone the pRb2 cDNA sequence. Primer A is an oligonucleotide containing 18 nucleotides coding for the amino acid sequence Phe-Tyr- Lys-Val-Ile-Glu (SEQ ID NO:3) . The 5' end of primer A also contains nine nucleotides which form a BamHI re- striction site. Primer B is an oligonucleotide containing 18 nucleotides coding for the amino acid sequence Gln-Asp- Leu-His-Arg-Asp (SEQ ID NO:4) . The 5' end of primer B also contains nine nucleotides which form a HindiII restriction site. Each of the 18-mer primers A and B correspond to conserved portions in the pocket regions of pRb and pl07. The two restriction sites (BamHI for primer A, and HindiII for primer B) were used to conveniently subclone the amplified PCR fragments into a commercially available vector (pBluescript, Stratagene, La Jolla, CA) . The PCR product was used as a probe for the screening of cDNA libraries from human 293 and HeLa cells. From the screening, several positive clones were identified. These clones were sequenced and analyzed for a clone containing full length cDNA. One of the HeLa cDNA clones contained a puta¬ tive initiation codon which is compatible with the Kozak initiation sequence (Kozak, J. Mol . Biol .. 196 947-950 (1987) . This clone showed a unique open reading frame ending in a termination codon 3,249 base pairs downstream (see SEQ ID NO:l) . The complete sequence included 55 base pairs upstream of the open reading frame which did not contain any putative initiation site, and a 3' non- coding region ending in a poly A tail. The open reading frame encoded a polypeptide of 1,082 amino acids (SEQ ID NO:2) with a predicted molecular mass of approximately 120 kD. This cDNA clone was designated rb2 and, hence, the encoded protein pRb2. The sequence of protein pRb2 (SEQ ID NO:2) as compared to the pRb and pi07 protein sequences shows a high level of identity, 53% with respect to pl07, and 32% with respect to pRb. This suggests a closer relationship of pRb2 to pl07. A partial comparisons of the amino acid sequences of these three proteins shows that the pocket region is clearly conserved in pRb2, mainly at the level of the domains A and B. This suggests that pRb2 has properties similar to pRb and pl07, such as the formation of cell cycle-associated protein complexes which are known to occur via the pocket region. This suggests that pRb2 would be involved in the cell cycle machinery. Moreover, the high identities found in the C and N- terminal portions between pRb2 and pl07 suggest a role for these regions in a function of pl07 and pRb2 which may differentiate them from pRb. The pRb2 cDNA clone was transcribed into an RNA segment in vitro by a T7 RNA polymerase capping reaction on the linearized pBluescript-pRb2. The resulting tran¬ scription product (RNA segment) was extracted with a phenol/chloroform solution and precipitated in an ethanol solution. The transcription product was used as a sub¬ strate for in vitro translation into a protein by using a rabbit reticulocyte lysate (Promega, Biotec, Madison, WI) and 35 S-methionine as a radioactive label (Pelham et. a______, Eur. J. Biochem. , 67 248-256 (1976)) . Several truncated forms of the protein were produced. The largest pRb2 protein form migrated to approximately 120 kD by SDS-PAGE. The most prominent of the bands corre¬ sponds to a protein form which migrated to around 90 kD, and a third protein form was found to migrate to 85 kD. After isolation, the 120 kD pRb2 protein and its 90 kD and 85 kD truncated forms were tested for ElA protein binding properties. The ElA binding results were compared to the ElA binding properties of the pRb and pl07 proteins. Both pRb and pl07 proteins bind to the adenovirus ElA protein through their respective pocket regions. Demonstration of ElA protein binding by the pRb2 protein would indicate that the latter protein has a key role in the regulation of cell division. The ElA inding capacity of pRb2 was thus determined as follows. Wild type and mutant forms of the ElA protein were obtained. The nature of the mutations are set forth in Figure 1. A binding assay was performed to test the binding of the wild type and mutant ElA proteins to in vitro-translated pRb2 protein (120 kD and truncated 90 kD and 85 kD proteins) precleared of translation solu¬ tion. Each of the three in vitro translated main forms of the pRb2 protein bound to ElA. An ElA deletion mutation involving the N-ter- minal portion of ElA (dl2-36) did not affect binding of the pRb2 to ElA. The binding of pRb2 was also not affected by deletion mutations involving the transforming domain 1 of ElA (ElA mutants dl38-67 and dl73-120) . This suggests that binding of pRb2 to ElA does not take place via these regions of ElA. However, the ability of the pRb2 protein to bind was almost completely abolished when an ElA mutant protein containing a double point mutation in the transforming domain 2 of ElA was used (ElA mutant pm928/961, in which Cys was substituted for Gly at position 124, and Lys for Glu at position 135) . There- fore, the transforming domain 2 of ElA is required for binding to pRb2. This suggests that the ElA-binding capacity of pRb2 is involved, at least in part, in the transforming activity of ElA. Although the pRb2 protein is similar in molecular weight to the pl30 protein and they both have similar binding profiles to ElA wild type and mutant proteins the two proteins are not identical. The pl30 protein is phosphorylated on Ser and Thr residues while pRb2 is unphosphorylated. Moreover, pl30 exists in more than one phosphorylated form. A cloning vector designated as pBluescript-pRb2 which contained the pRb2 cDNA was deposited with the American Type Culture Collection, Rockville, MD on August 11, 1993, and was given the accession number 75521. An E. coli bacterial strain designated as E. coli pBluescript-pRb2, which contained a plasmid containing the pRb2 cDNA was deposited with the ATCC, Rockville, MD on August 11, 1993 and given the accession number 69383. It is well within the skill of those in the genetic engineering art to use the nucleotide sequence of SEQ ID N0:1 or related sequences encoding for the pRb2 protein of the present invention to produce pRb2 protein via microbial processes. Using the nucleotide sequence of SEQ ID NO:l to produce pRb2 is made easier for one of ordinary skill by utilizing the pBluescript-pRb2 cloning vector according to the invention. Fusing the nucleotide sequences encoding for the pRb2 protein into an expression vector and trans- forming or transfecting into hosts, either eukaryotic (yeast or mammalian cells) or prokaryotic (bacterial cells) , are standard procedures used in producing other well-known proteins, e.g. , insulin, interferons, human growth hormone, and the like. Similar procedures, or obvious modifications thereof, can be employed to prepare pRb2 proteins by microbial means or mammalian tissue- culture technology in accord with the subject invention. The nucleic acid molecules that encode pRb2 protein may be inserted into known vectors for use in standard recombinant DNA techniques for producing recom- binant pRb2 protein. Standard recombinant DNA techniques include those techniques as described by Sambrook et. al. , Eds., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, 1989 and by Ausubel et al. , Current Protocols in Molecular Biology. J. Wiley & Sons, New York, NY (1991) . The vectors may be circular or non-circular. The host nay be prokaryotic or eukaryotic. The preferred prokaryotic host comprises E. coli. Preferred eukaryotic hosts include yeasts, insect and mammalian cells. Preferred mammalian cells include, primate cells such monkey cells transformed by simian viruses (e.g., COS cells), human cells, and Chinese ham¬ ster ovary (CHO) cells. Those of ordinary skill in the art will appreciate the fact that the cDNA fragment set forth in SEQ ID NO:l is only one DNA segment coding for pRb2. Other equivalent DNA segments of substantial similarity will immediately be envisioned which will code for pRb2. The present invention also includes such equivalent DNA sequences. Moreover, substitution of equivalent amino acids in SEQ ID NO:2 would not be expected to affect the pRb2 protein's activity. These amino acid substitutions would be envisioned by those of ordinary skill in the art. Such equivalent amino acid sequences are also included within the present invention. The following non-limited examples are provided to illustrate the invention. Examples EXAMPLE 1 : Obtaining pRb2 cDNA and Amino Acid Sequences A. Synthesis of PCR Primers Primers A and B were synthesized using standard oligonucleotide synthesis techniques and purified. Primer A contained 18 nucleotides coding for the polypeptide sequence according to SEQ ID NO:3. The 5 ' -end was of nine additional nucleotides that formed a BamHI restriction site. Primer B contained 18 nucleo¬ tides coding for the polypeptide sequence according to SEQ ID NO:4. The 5 ' -end was of nine additional nucleo¬ tides which formed a Hindlll restriction site. B. PCR Amplification of a Human 293 cDNA Library A lambda-ZAPII cDNA library was obtained from reverse transcription of RNA from human 293 cells using standard techniques. The cDNA library was amplified via PCR with Primers A and B from Example 1A above. The PCR was performed using a GeneAmp™ kit (Perkin Elmer Cetus, Norwalk, CN) according to the instructions of the manufacturer. Briefly, thirty cycles including a one minute denaturization at 94°C, one minute annealing at 37°C, and two minutes extension at 68°C, were followed by fifteen minute extension. This resulted in PCR amp¬ lification of a lkb fragment in addition to pRb and pl07 segments. C. Subcloning and Nucleotide Sequencing the lkb Fragment The amplified lkb fragment was subcloned into a pBluescript vector (Stratagene) . After subcloning, nucleotide sequencing was performed using the dideoxy method of the Sequenase kit (United States Biochemicals) . The nucleotide sequence of the lkb fragment revealed some homology with pRb and pl07 cDNAs. D. Probing cDNA Libraries From 293 and HeLa Cells The lkb fragment was utilized as a probe to screen additional cDNA libraries. Lambda-ZAPII cDNA libraries from human 293 and HeLa cells (Stratagene) , respectively were screened using the lkb fragment labeled with c-- 32 P-CTP by the random primer method (Boehringer Mannheim) . Briefly, lambda-ZAP phage was adsorbed to Escherichia coli BB4 strain bacteria and plated in agar medium. Nitrocellulose filters were hybridized to the PCR probe in a high stringency protocol which included the pre-hybridization mixture: 5x SSPE, lOx Denhardt's solution, 150 μg/ml herring sperm DNA, 50% formamide and 2% SDS; a hybridization mixture adding 10 6 cpm/ml of the lkb PCR probe to the pre-hybridization mixture; and, three washes of twenty minutes each at 42°C with 0.2x SSC and 0.1% SDS. E. Analyzing the Positive Clones From the Probing From the probing procedures of Example ID sev¬ eral positive clones were located. In vivo excision (Stratagene) was performed on the several positive Lambda-ZAP clones. pBluescript vectors containing cDNA clones were obtained. The cDNA clones were reproduced and nucleotide sequencing of each cDNA clone was per¬ formed as described above in Example 1C. The sequencing results analyzed for the full length cDNA including the lkb fragment. One of the HeLa cDNA clones contained a puta¬ tive initiation codon which was compatible with the Kozak initiation sequence. This clone showed a unique open reading frame ending in a termination codon 3,249 base pairs downstream (see SEQ ID NO:l) . The complete se¬ quence included 55 base pairs upstream of the open reading frame which did not contain any putative initi¬ ation site, and a 3' non-coding region ending in a poly A tail. The open reading frame encoded a polypeptide of 1,082 amino acids (SEQ ID NO:2) with a predicted mo- lecular mass of approximately 120 kD. This cDNA clone was designated rb2 and, hence, the encoded protein pRb2. The pBluescript vector cloned with the pRb2 gene was designated as pBluescript-pRb2. The sequence of protein pRb2 (SEQ ID NO:2, which is derived from the corresponding cDNA sequence) as compared to pRb and pi07 protein sequences showed a high level of identity, 53% with respect to pl07, and 32% with respect to pRb. This suggests a closer relationship of pRb2 to pl07. Partial comparisons of these three pro- tein sequences show that the pocket region is clearly conserved in pRb2, mainly at the level of the domains A and B. EXAMPLE 2: ElA Binding of In Vitro-Translated pRb2 A. Transcription and Translation of pBluescript-pRb2 The Example IE pBluescript-pRb2 cDNA clone was transcribed into an RNA segment in vitro by a T7 RNA polymerase capping reaction on the linearized pBluescript-pRb2. The resulting transcription product (RNA segment) was extracted with a phenol/chloroform solution and precipitated in an ethanol solution. The transcription product RNA segment was used as a substrate for in vitro translation into a protein by using a rabbit reticulocyte lysate (Promega, Biotec, Madison, WI) and 35 S-methionine as a radioactive label (Pelham et al. , Eur. J. Biochem. , 67 248-256 (1976) ) . Several truncated forms of the protein were produced. The largest form migrated to approximately 120 kD by SDS- PAGE. The most prominent of these bands migrated to around 90 kD, and a third one was found to migrate to 85 kD. B. Obtaining Wild-Type and Mutant ElA Proteins Wild type and mutant forms of the ElA protein were obtained. The mutant forms were pm928/961, dl2-36, dl38-67 and dl73-120. The nature of the mutations are set forth in Figure 1. The wild type and mutant forms of ElA were sub-cloned into pGEX-2T and expressed in E. coli as GST-fusion proteins. The ElA proteins were then isolated from E. coli cultures by standard techniques. C. Binding Assay for pRb2 and ElA Proteins A binding assay was performed to test the binding of the wild type and mutant ElA proteins of Example 2B with the in vitro-translated pRb2 proteins of Example 2A precleared of translation solution. The pRb2 protein (120 kD, 90 kD, and 85 kD) was precleared with glutathione-sepharose and GST-glutathione-sepharose beads in NETN buffer containing 1 mM DTT, 1 mM PMSF, and 10 μg/ml leupeptine, at 4°C. Two μg of each ElA protein were incubated with precleared pRb2 for one hour at 4°C, and glutathione- sepharose beads were added and incubated for an additional hour. Proteins were resolved using SDS-PAGE according to standard protocols and a Fuji phosphoimage analyzer system was used to develop the protein signal. The results of the binding assay are set forth in Figure 2, which is an SDS-PAGE showing binding to wild-type ElA (lane 2) and to ElA mutant constructs: dl2- 36 (lane 3), dl38-67 (lane 4), dl73-120 (lane 5), and pm928/961 (lane 6) . GST with no ElA fused was included as a control (lane 1) . The in vitro-translated product resulting from a rabbit reticulocyte translation reaction with no exogenous RNA was included as a control, which did not give any signal (not shown) . Each of the three in vitro translated main forms of the pRb2 protein (120 kD, 90 kD, and 80 kD) bound to ElA. A deletion mutation involving the N- terminal portion of ElA (dl2-36) did not affect pRb2 binding. ElA binding to pRb2 was not affected by dl38-67 or dl73-120 deletion mutations, both involving the transforming domain 1 of ElA. This suggests that binding of pRb2 to ElA does not take place in these regions. However, binding was almost completely abolished when an ElA-fusion protein containing a double point mutation in the transforming domain 2 of ElA was used (ElA mutant pm928/961, in which Cys was substituted for Gly at posi¬ tion 124, and Lys for Glu at position 135) . Therefore, the transforming domain 2 of ElA is required for binding to pRb2. This suggests that the ElA-binding capacity of pRb2 is involved, at least in part, in the transforming activity of ElA. For comparative purposes the pRb and pi07 proteins were obtained and a binding assay performed with the wild type and mutant ElA proteins. The pRb2 protein showed similar binding characteristics to the pRb and pl07 proteins. Since the pRb2 protein binds the ElA protein in manner similar to the pRb protein, the pRb2 protein is a useful diagnostic tool for identifying cells infect¬ ed with adenovirus ElA, or a related DNA virus producing oncoproteins related to the ElA protein. Because of the ElA binding capacity of pRb2, the protein may be adminis¬ tered to cells infected with adenovirus ElA, where it may act as a cell growth suppressor to reverse the effects of the ElA oncoprotein. This reversal of ElA protein effects could restore the balance of cell growth in a retino-blastoma cancer tumor. Thus, pRb2 is believed useful as tumor suppressor agent, for treating cancers such as retinoblastoma interocular cancer. Further, pRb2 may be a useful research tool for binding and identifying other DNA tumor virus oncoproteins which have sequences related to the ElA protein. All references cited with respect to synthetic,, preparative and analytical procedures are incorporated herein by reference. The present invention may be embodied in other specific forms without departing from the spirit or es¬ sential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT(S) : Antonio Giordano (ii) TITLE OF INVENTION: "TUMOR SUPPRESSOR PROTEIN PRB2, RELATED GENE PRODUCTS, AND DNA ENCODING THEREFOR" (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Seidel, Gonda, Lavorgna & Monaco, P.C. (B) STREET: Two Penn Center, Suite 1800 (C) CITY: Philadelphia (D) STATE: Pennsylvania (E) COUNTRY: U.S.A. (F) ZIP: 19102 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: August 12, 1994 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/106,493 (B) FILING DATE: August 12, 1993 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Daniel A. Monaco (B) REGISTRATION NUMBER: 30,480 (C) REFERENCE/DOCKET NUMBER: 6056-188 PC (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (215) 568-8383 (B) TELEFAX: (215) 568-5549 (2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3249 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGGACGAGG CGGCGCGGGC CGAGGCCTGG GACAGCTACC GCAGCATGAG 50 CGAAAGCTAC ACGCTGGAGG GAAATGATCT TCATTGGTTA GCATGTGCCT 100 TATATGTGGC TTGCAGAAAA TCTGTTCCAA CTGTAAGCAA AGGGACAGTG 150 GAAGGAAACT ATGTATCTTT AACTAGAATC CTGAAATGTT CAGAGCAGAG 200 CTTAATCGAA TTTTTTAATA AGATGAAGAA GTGGGAAGAC ATGGCAAATC 250 TACCCCCACA TTTCAGAGAA CGTACTGAGA GATTAGAAAG AAACTTCACT 300 GTTTCTGCTG TAATTTTTAA GAAATATGAA CCCATTTTTC AGGACATCTT 350 TAAATACCCT CAAGAGGAGC AACCTCGTCA GCAGCGAGGA AGGAAACAGC 400 GGCGACAGCC CTGTACTGTG TCTGAAATTT TCCATTTTTG TTGGATGCTT 450 TTTATATATG CAAAAGGTAA TTTCCCCATG ATTAGTGATG ATTTGGTCAA 500 TTCTTATCAC CTGCTGCTGT GTGCTTTGGA CTTAGTTTAT GGAAATGCAC 550 TTCAGTGTTC TAATCGTAAA GAACTTGTGA ACCCTAATTT TAAAGGCTTA 600 TCTGAAGATT TTCATGCTAA AGATTCTAAA CCTTCCTCTG ACCCCCCTTG 650 TATCATTGAG AAACTGTGTT CCTTACATGA TGGCCTAGTT TTGGAAGCAA 700 AGGGGATAAA GGAACATTTC TGGAAACCCT ATATTAGGAA ACTTTATGAA 750 AAAAAGCTCC TTAAGGGAAA AGAAGAAAAT CTCACTGGGT TTCTAGAACC 800 TGGGAACTTT GGAGAGAGTT TTAAAGCCAT CAATAAGGCC TATGAGGAGT 850 ATGTTTTATC TGTTGGGAAT TTAGATGAGC GGATATTTCT TGGAGAGGAT 900 GCTGAGGAGG AAATTGGGAC TCTCTCAAGG TGTCTGAACG CTGGTTCAGG 950 AACAGAGACT GCTGAAAGGG TGCAGATGAA AAACATCTTA CAGCAGCATT 1000 TTGACAAGTC CAAAGCACTT AGAATCTCCA CACCACTAAC TGGTGTTAGG 1050 TACATTAAGG AGAATAGCCC TTGTGTGACT CCAGTTTCTA CAGCTACGCA 1100 TAGCTTGAGT CGTCTTCACA CCATGCTGAC AGGCCTCAGG AATGCACCAA 1150 GTGAGAAACT GGAACAGATT CTCAGGACAT GTTCCAGAGA TCCAACCCAG 1200 GCTATTGCTA ACAGACTGAA AGAAATGTTT GAAATATATT CTCAGCATTT 1250 CCAGCCAGAC GAGGATTTCA GTAATTGTGC TAAAGAAATT GCCAGCAAAC 1300 ATTTTCGTTT TGCGGAGATG CTTTACTATA AAGTATTAGA ATCTGTTATT 1350 GAGCAGGAAC AAAAAAGACT AGGAGACATG GATTTATCTG GTATTCTGGA 1400 ACAAGATGCG TTCCACAGAT CTCTCTTGGC CTGCTGCCTT GAGGTCGTCA 1450 CTTTTTCTTA TAAGCCTCCT GGGAATTTTC CATTTATTAC TGAAATATTT 1500 GATGTGCCTC TTTATCATTT TTATAAGGTG ATAGAAGTAT TCATTAGAGC 1550 AGAAGATGGC CTTTGTAGAG AGGTGGTAAA ACACCTTAAT CAGATTGAAG 1600 AACAGATCTT AGATCATTTG GCATGGAAAC CAGAGTCTCC ACTCTGGGAA 1650 AAAATTAGAG ACAATGAAAA CAGAGTTCCT ACATGTGAAG AGGTCATGCC 1700 ACCTCAGAAC CTGGAAAGGG CAGATGAAAT TTGCATTGCT GGCTCCCCTT 1750 TGACTCCCAG AAGGGTGACT GAAGTTCGTG CTGATACTGG AGGACTTGGA 1800 AGGAGCATAA CATCTCCAAC CACATTATAC GATAGGTACA GCTCCCCACC 1850 AGCCAGCACT ACCAGAAGGC GGCTATTTGT TGAGAATGAT AGCCCCTCTG 1900 ATGGAGGGAC ACCTGGGCGG ATGCCCCCAC AGCCCCTAGT CAATGCTGTC 1950 CCTGTGCAGA ATGTATCTGG GGAGACTGTT TCTGTCACAC CAGTTCCTGG 2000 ACAGACTTTG GTCACCATGG CAACCGCCAC TGTCACAGCC AACAATGGGC 2050 AAACGGTAAC CATTCCTGTG CAAGGTATTG CCAATGAAAA TGGAGGGATA 2100 ACATTCTTCC CTGTCCAAGT CAATGTTGGG GGGCAGGCAC AAGCTGTGAC 2150 AGGCTCCATC CAGCCCCTCA GTGCTCAGGC CCTGGCTGGA AGTCTGAGCT 2200 CTCAACAGGT GACAGGAACA ACTTTGCAAG TCCCTGGTCA AGTGGCCATT 2250 CAACAGATTT CCCCAGGTGG CCAACAGCAG AAGCAAGGCC AGTCTGTAAC 2300 CAGCAGTAGT AATAGACCCA GGAAGACCAG CTCTTTATCG CTTTTCTTTA 2350 GAAAGGTATA CCATTTAGCA GCTGTCCGCC TTCGGGATCT CTGTGCCAAA 2400 CTAGATATTT CAGATGAATT GAGGAAAAAA ATCTGGACCT GCTTTGAATT 2450 CTCCATAATT CAGTGTCCTG AACTTATGAT GGACAGACAT CTGGACCAGT 2500 TATTAATGTG TGCCATTTAT GTGATGGCAA AGGTCACAAA AGAAGATAAG 2550 TCCTTCCAGA ACATTATGCG TTGTTATAGG ACTCAGCCGC AGGCCCGGAG 2600 CCAGGTGTAT AGAAGTGTTT TGATAAAAGG GAAAAGAAAA AGAAGAAATT 2650 CTGGCAGCAG TGATAGCAGA AGCCATCAGA ATTCTCCAAC AGAACTAAAC 2700 AAAGATAGAA CCAGTAGAGA CTCCAGTCCA GTTATGAGGT CAAGCAGCAC 2750 CTTGCCAGTT CCACAGCCCA GCAGTGCTCC TCCCACACCT ACTCGCCTCA 2800 CAGGTGCCAA CAGTGACATG GAAGAAGAGG AGAGGGGAGA CCTCATTCAG 2850 TTCTACAACA ACATCTACAT CAAACAGATT AAGACATTTG CCATGAAGTA 2900 CTCACAGGCA AATATGGATG CTCCCCCACT CTCTCCCTAT CCATTTGTAA 2950 GAACAGGCTC CCCTCGCCGA ATACAGTTGT CTCAAAATCA TCCTGTCTAC 3000 ATTTCCCCAC ATAAAAATGA AACAATGCTT TCTCCTCGAG AAAAGATTTT 3050 CTATTACTTC AGCAACAGTC CTTCAAAGAG ACTGAGAGAA ATTAATAGTA 3100 TGATACGCAC AGGAGAAACT CCTACTAAAA AGAGAGGAAT TCTTTTGGAA 3150 GATGGAAGTG AATCACCTGC AAAAAGAATT TGCCCAGAAA ATCATTCTGC 3200 CTTATTACGC CGTCTCCAAG ATGTAGCTAA TGACCGTGGT TCCCACTGA 3249 (3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1082 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Glu Ala Ala Arg Ala Glu Ala Trp Asp Ser Tyr Arg Ser 5 10 15 Met Ser Glu Ser Tyr Thr Leu Glu Gly Asn Asp Leu His Trp Leu 20 25 30 Ala Cys Ala Leu Tyr Val Ala Cys Arg Lys Ser Val Pro Thr Val 35 40 45 Ser Lys Gly Thr Val Glu Gly Asn Tyr Val Ser Leu Thr Arg lie 50 55 60 Leu Lys Cys Ser Glu Gin Ser Leu lie Glu Phe Phe Asn Lys Met 65 70 75 Lys Lys Trp Glu Asp Met Ala Asn Leu Pro Pro His Phe Arg Glu 80 85 90 Arg Thr Glu Arg Leu Glu Arg Asn Phe Thr Val Ser Ala Val He 95 100 105 Phe Lys Lys Tyr Glu Pro He Phe Gin Asp He Phe Lys Tyr Pro 110 115 120 Gin Glu Glu Gin Pro Arg Gin Gin Arg Gly Arg Lys Gin Arg Arg 125 130 135 Gin Pro Cys Thr Val Ser Glu He Phe His Phe Cys Trp Met Leu 140 145 150 Phe He Tyr Ala Lys Gly Asn Phe Pro Met He Ser Asp Asp Leu 155 160 165 Val Asn Ser Tyr His Leu Leu Leu Cys Ala Leu Asp Leu Val Tyr 170 175 180 Gly Asn Ala Leu Gin Cys Ser Asn Arg Lys Glu Leu Val Asn Pro 185 190 195 Asn Phe Lys Gly Leu Ser Glu Asp Phe His Ala Lys Asp Ser Lys 200 205 210 Pro Ser Ser Asp Pro Pro Cys He He Glu Lys Leu Cys Ser Leu 215 220 225 His Asp Gly Leu Val Leu Glu Ala Lys Gly He Lys Glu His Phe 230 235 240 Trp Lys Pro Tyr He Arg Lys Leu Tyr Glu Lys Lys Leu Leu Lys 245 250 255 Gly Lys Glu Glu Asn Leu Thr Gly Phe Leu Glu Pro Gly Asn Phe 260 265 270 Gly Glu Ser Phe Lys Ala He Asn Lys Ala Tyr Glu Glu Tyr Val 275 280 285 Leu Ser Val Gly Asn Leu Asp Glu Arg He Phe Leu Gly Glu Asp 290 295 300 Ala Glu Glu Glu He Gly Thr Leu Ser Arg Cys Leu Asn Ala Gly 305 310 315 Ser Gly Thr Glu Thr Ala Glu Arg Val Gin Met Lys Asn He Leu 320 325 330 Gin Gin His Phe Asp Lys Ser Lys Ala Leu Arg He Ser Thr Pro 335 340 345 Leu Thr Gly Val Arg Tyr He Lys Glu Asn Ser Pro Cys Val Thr 350 355 360 Pro Val Ser Thr Ala Thr His Ser Leu Ser Arg Leu His Thr Met 365 370 375 Leu Thr Gly Leu Arg Asn Ala Pro Ser Glu Lys Leu Glu Gin He 380 385 390 Leu Arg Thr Cys Ser Arg Asp Pro Thr Gin Ala He Ala Asn Arg 395 400 405 Leu Lys Glu Met Phe Glu He Tyr Ser Gin His Phe Gin Pro Asp 410 415 420 Glu Asp Phe Ser Asn Cys Ala Lys Glu He Ala Ser Lys His Phe 425 430 435 Arg Phe Ala Glu Met Leu Tyr Tyr Lys Val Leu Glu Ser Val He 440 445 450 Glu Gin Glu Gin Lys Arg Leu Gly Asp Met Asp Leu Ser Gly He 455 460 465 Leu Glu Gin Asp Ala Phe His Arg Ser Leu Leu Ala Cys Cys Leu 470 475 480 Glu Val Val Thr Phe Ser Tyr Lys Pro Pro Gly Asn Phe Pro Phe 485 490 495 He Thr Glu He Phe Asp Val Pro Leu Tyr His Phe Tyr Lys Val 500 505 510 He Glu Val Phe He Arg Ala Glu Asp Gly Leu Cys Arg Glu Val 515 520 525 Val Lys His Leu Asn Gin He Glu Glu Gin He Leu Asp His Leu 530 535 540 Ala Trp Lys Pro Glu Ser Pro Leu Trp Glu Lys He Arg Asp Asn 545 550 555 Glu Asn Arg Val Pro Thr Cys Glu Glu Val Met Pro Pro Gin Asn 560 565 570 Leu Glu Arg Ala Asp Glu He Cys He Ala Gly Ser Pro Leu Thr 575 580 585 Pro Arg Arg Val Thr Glu Val Arg Ala Asp Thr Gly Gly Leu Gly 590 595 600 Arg Ser He Thr Ser Pro Thr Thr Leu Tyr Asp Arg Tyr Ser Ser 605 610 615 Pro Pro Ala Ser Thr Thr Arg Arg Arg Leu Phe Val Glu Asn Asp 620 625 630 Ser Pro Ser Asp Gly Gly Thr Pro Gly Arg Met Pro Pro Gin Pro 635 640 645 Leu Val Asn Ala Val Pro Val Gin Asn Val Ser Gly Glu Thr Val 650 655 660 Ser Val Thr Pro Val Pro Gly Gin Thr Leu Val Thr Met Ala Thr 665 670 675 Ala Thr Val Thr Ala Asn Asn Gly Gin Thr Val Thr He Pro Val 680 685 690 Gin Gly He Ala Asn Glu Asn Gly Gly He Thr Phe Phe Pro Val 695 700 705 Gin Val Asn Val Gly Gly Gin Ala Gin Ala Val Thr Gly Ser He 710 715 720 Gin Pro Leu Ser Ala Gin Ala Leu Ala Gly Ser Leu Ser Ser Gin 725 730 735 Gin Val Thr Gly Thr Thr Leu Gin Val Pro Gly Gin Val Ala He 740 745 750 Gin Gin He Ser Pro Gly Gly Gin Gin Gin Lys Gin Gly Gin Ser 755 760 765 Val Thr Ser Ser Ser Asn Arg Pro Arg Lys Thr Ser Ser Leu Ser 770 775 780 Leu Phe Phe Arg Lys Val Tyr His Leu Ala Ala Val Arg Leu Arg 785 790 795 Asp Leu Cys Ala Lys Leu Asp He Ser Asp Glu Leu Arg Lys Lys 800 805 810 He Trp Thr Cys Phe Glu Phe Ser He He Gin Cys Pro Glu Leu 815 820 825 Met Met Asp Arg His Leu Asp Gin Leu Leu Met Cys Ala He Tyr 830 835 840 Val Met Ala Lys Val Thr Lys Glu Asp Lys Ser Phe Gin Asn He 845 850 855 Met Arg Cys Tyr Arg Thr Gin Pro Gin Ala Arg Ser Gin Val Tyr 860 865 870 Arg Ser Val Leu He Lys Gly Lys Arg Lys Arg Arg Asn Ser Gly 875 880 885 Ser Ser Asp Ser Arg Ser His Gin Asn Ser Pro Thr Glu Leu Asn 890 895 900 Lys Asp Arg Thr Ser Arg Asp Ser Ser Pro Val Met Arg Ser Ser 905 910 915 Ser Thr Leu Pro Val Pro Gin Pro Ser Ser Ala Pro Pro Thr Pro 920 925 930 Thr Arg Leu Thr Gly Ala Asn Ser Asp Met Glu Glu Glu Glu Arg 935 940 945 Gly Asp Leu He Gin Phe Tyr Asn Asn He Tyr He Lys Gin He 950 955 960 Lys Thr Phe Ala Met Lys Tyr Ser Gin Ala Asn Met Asp Ala Pro 965 970 975 Pro Leu Ser Pro Tyr Pro Phe Val Arg Thr Gly Ser Pro Arg Arg 980 985 990 He Gin Leu Ser Gin Asn His Pro Val Tyr He Ser Pro His Lys 995 1000 1005 Asn Glu Thr Met Leu Ser Pro Arg Glu Lys He Phe Tyr Tyr Phe 1010 1015 1020 Ser Asn Ser Pro Ser Lys Arg Leu Arg Glu He Asn Ser Met He 1025 1030 1035 Arg Thr Gly Glu Thr Pro Thr Lys Lys Arg Gly He Leu Leu Glu 1040 1045 1050 Asp Gly Ser Glu Ser Pro Ala Lys Arg He Cys Pro Glu Asn His 1055 1060 1065 Ser Ala Leu Leu Arg Arg Leu Gin Asp Val Ala Asn Asp Arg Gly 1070 1075 1080 Ser His (4 ) INFORMATION FOR SEQ ID NO : 3 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 6 amino acids (B) TYPE : amino acid (D) TOPOLOGY : linear (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 3 : Phe Tyr Lys Val He Glu 5 (5) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gin Asp Leu His Arg Asp 5

Claims: CLAIMS 1. A recombinant DNA cloning vehicle comprising a DNA sequence comprising the human pRb2 gene DNA sequence. 2. A cloning vehicle according to claim 1 comprising a DNA coding for a protein having the amino acid sequence SEQ ID NO:2. 3. A cloning vehicle according to claim 2 wherein the DNA sequence comprises the DNA sequence SEQ ID NO:l. 4. A cloning vehicle according to claim 3, which is a pBluescript-pRb2 vehicle. 5. A cloning vehicle according to claim 4, which is a pBluescript-pRb2 vehicle having ATCC No. 75521. 6. Essentially pure DNA coding for human pRb2, having the nucleotide sequence SEQ ID NO:l. 7. Essentially pure DNA according to claim 6 having a DNA sequence coding for a protein having the amino acid sequence SEQ ID NO:2. 8. Essentially pure DNA according to claim 7 having a DNA sequence coding for a protein having the amino acid sequence SEQ ID NO:2. 9. An isolated protein which is human pRB2. 10. A protein according to claim 9 having the amino acid sequence SEQ ID NO:2. 11. A protein according to claim 10, which is not phosphorylated. 12. A host transformed by a cloning vehicle according to claim 1. 13. A host transformed by a cloning vehicle according to claim 2. 14. A host transformed by a cloning vehicle according to claim 3. 15. A transformed host according to claim 12 which is a cell line. 16. A transformed host according to claim 13 which is a cell line. 17. A transformed host according to claim 14 which is a cell line. 18. A host cell line according to claim 15 which is ATCC No. 69383. 19. A process for preparing human pRb2 protein which comprises culturing a host cell hosting a cloning vehicle according to claim 1.

Inventors: GIORDANO ANTONIO, GIORDANO, ANTONIO
Applicants: UNIV TEMPLE, TEMPLE UNIVERSITY OF THE COMMONWEALTH SYSTEM OF HI
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, C07H21, C07K14, C12N1, C12N15, C12P21, C12R1